This program explores innate immune, pro-inflammatory, and signaling functions of NOX family NADPH oxidases. The current research is focused on non-phagocytic oxidases (NOX1, NOX4, DUOX1, DUOX2) expressed primarily in epithelial cells, notably on mucosal surfaces (lung and gastrointestinal tract), in liver, kidney, thyroid and exocrine glands (salivary, mammary), and in vascular tissues. Deliberate reactive oxygen species (ROS) production by these enzymes can affect cell migration, proliferation, tumor invasiveness and metastasis, cell differentiation, senescence, apoptosis, oxygen sensing, extracellular matrix and thyroid hormone biosynthesis, microbial killing, and responses to cytokines, growth factors, hormones and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In 2017, we explored the functions of several epithelial NADPH oxidases in cell migration, metastasis, proliferation, barrier functions and responses to microbes (pathogenic and nonpathogenic) on mucosal surfaces: 1) The mechanistic basis for NOX4 induction by common tumor-associated TP53 mutations was studied in several tumor types. 2) Roles for DUOX1 in innate immune responses of respiratory epithelium to influenza A virus infection were examined in DUOX1-deficient mice. 3) The inflammatory functions of intestinal epithelial NOX enzymes were examined in several mouse gene knockout models. Earlier, we showed NOX4 is uniquely induced by transformed growth factor-beta (TGF-beta) and is involved in the epithelial-to-mesenchymal transition (EMT), consistent with proposed roles for NOX4 in the development of fibrotic disease in lung, liver and kidney. Recently, we showed wild-type (WT) and mutant (mut) forms of p53 have opposing effects on TGF-beta induction of NOX4 through its transcriptional regulator, SMAD3: p53-WT suppresses TGF/SMAD3-induced NOX4, ROS and tumor cell migration, whereas tumor-associated p53-mut proteins enhance NOX4 expression and metastatic cell migration. To establish the broad significance of this model suggesting NOX4 is a driver of metastatic cell activity, we surveyed data from approximately 2000 primary tumor specimens in The Cancer Genome Atlas (TCGA) and confirmed higher NOX4 expression in tumors with several common p53 hotspot mutations also observed in Li-Fraumeni syndrome. NOX4 expression is particularly high in breast, pancreatic and head and neck primary tumors with mut-p53, which correlates with EMT markers that predict invasive and migratory cell phenotypes. Several approaches were used to establish direct NOX4 involvement in cell migration downstream of mut-p53 signaling, including p53 silencing or overexpression, NOX4 silencing, or expression of dominant-negative NOX4 to inhibit tumor cell migration. We showed the convergence of TGF-beta/SMAD3 and p53 pathways regulating NOX4 occurs at two levels, through direct genetic and epigenetic regulatory mechanisms: 1) WT- and mut-p53 differentially interact with SMAD3 within complexes on two SMAD3-binding elements of the NOX4 gene and 2) WT and mut-p53 differentially regulate histone (H-4) acetylation within the NOX4 locus. In support of the latter mechanism, histone deacetylase inhibitors were shown to relieve the repressive effects of WT p53, whereas the NOX4-inducing effects of mut-p53 involve recruitment of the histone acetyltransferase, p300, to the NOX4 locus. Furthermore, wound closure assays demonstrated that p300 through NOX4 promotes TGF-/mut-p53-mediated tumor cell migration. These findings provide new insight into mut-p53 induction of metastatic cell migration involving epigenetic (histone acetylation-based) control of NOX4, and suggest novel pharmacological, genetic and epigenetic approaches targeting NOX4 may be used to inhibit metastasis of advanced tumors with TP53 mutations. Since our previous work showed several bacterial species trigger human respiratory cell DUOX1 activity and that the release of DUOX-derived hydrogen peroxide is sufficient to kill several airway pathogens, we examined the susceptibility of DUOX1 knockout mice to influenza A virus infection. In work with Helene Rosenberg (NIAID), we compared several indicators of disease pathogenesis in DUOX1-deficient and control mice infected with influenza virus (strain A/HK/68 (H3N2)) over the course of 7-10 days post-infection. No significant differences in overall weight loss, lung inflammatory cell infiltration or recoverable viral titers were observed between strains, suggesting that DUOX1 in mouse airways neither enhances nor suppresses the disease pathogenesis of influenza A infection in mice. Other studies sought to identify sources of ROS in gut epithelium and establish their roles in inflammatory disease (colitits). Mice deficient in glutathione peroxidase 1 and 2 (GPx1/2) exhibit spontaneous colitis, the severity of which depends on mouse strain background. Previous work with Fong-Fong Chu identified DUOX2 as a candidate source of excess ROS in colon epithelium within a glutathione peroxidase-deficiency associated colitis (gdac-1) locus, as it was linked to milder inflammation severity on congenic 129 GPx1/2 double knockout mice carrying the B6 gdac-1 locus. Here, we found that the absence of DUOX activators (DUOXA) needed to support intestinal DUOX2 alleviates ileocolitis symptoms in GPx1/2 knockout mice. In comparing DUOX2 activities of four non-synonymous single nucleotide polymorphic (SNP) variants between 129 and B6 strains, we found the 129 H627R SNP variant exhibits 7.6-fold higher activity than the B6 variant. Together, the results suggest DUOX2 activity exacerbates redox imbalance and disease severity in GPx1/2 KO mice, which can be attributed to the effects of the H627R SNP. Ongoing studies with Emilia Falcone (NIAID) are exploring causes of enhanced colitis susceptibility in p47phox-deficent mice by comparing colon epithelial ROS generation and NOX expression in several other mouse strains.